We aim to elucidate the molecular mechanisms that control the assembly of viruses with the ultimate goals of defining targets for antiviral compounds, characterizing viral antigenicity, and establishing precedents for understanding the assembly of macromolecular complexes in general. Our research focuses on the large-scale conformational changes that accompany capsid maturation and on the interaction of viruses with host cells. To this end, we pursue five subprojects. (1) Hepatitis B Virus Capsid Assembly. Background. We have been studying the HBV capsid protein which carries two major antigens ? ?core antigen? and ?e-antigen?. After our first studies showed that the protein forms dimers capable of self-assembly into particles of two different sizes and its fold is predominantly alpha-helical, in 1997, we calculated a cryo-EM density map at 0.9nm resolution, in which much of the secondary structure was visible, including the 4-helix bundle that forms the dimerization motif. Our subsequent research helped delineate the overall path of the polypeptide chain, which has since been confirmed by crystallography. We are now investigating the range of antigenic diversity (distinct epitopes simultaneously present) on the HBV capsid. Results. We have used cryo-EM of Fab-labelled capsids to study the capsid binding of a set of monoclonal antibodies with conformational epitopes which precludes mapping them by immunochemical methods. The availability of atomic models of the capsid and prototypic Fabs allows us to identify the sets of discontinuous peptides that make up these epitopes. In this way we have characterized the binding of three monoclonals. All have quite different epitopes. Conclusions. These observations imply that a considerable range of antigenic diversity is presented by the capsid spike ? a relatively simple motif of two helix-loop-helix elements. More broadly, this approach holds promise for elucidating other neutralizing and diagnostic viral epitopes, most of which are conformational. (2) Capsid Assembly and Maturation of Herpesviruses. Herpesviruses constitute an extensive family of large, complex, DNA viruses. Eight members cause diseases in humans, including skin diseases. We have studied herpesvirus capsid assembly for several family members, including herpes simplex (HSV), and cytomegalovirus (CMV). Initially we defined the molecular anatomy of the capsid and then characterized the roles of its six major proteins in assembly. These roles are distinct from any other known family of animal viruses but share numerous points of resemblance with DNA bacteriophages. One major finding was our discovery, in 1996, of the HSV procapsid - a normally short-lived precursor that differs radically from the mature capsid in structure, stability, and composition. Maturation of the procapsid is controlled by the viral protease and is a potential target for antiviral drugs. We have been investigating this process of time-resolved cryo-EM. Results and Conclusions. Procapsids were isolated and allowed to mature spontaneously over a 48-hour period. Samples were taken for cryo-EM imaging at regular intervals. The resulting data set (5000 particles) was classified into 17 discreet morphological types which were assigned as a time-ordered series from their waxing and waning at successive time-points. The transition involves a single major cooperative change about stage 8, preceeded by small adjustments of the original procapsid and succeeded by minor ?aftershocks?. This experiment shows that it is feasible to monitor dynamic processes of macromolecular complexes by cryo-EM, using image classification to identify distinct transition states. Procapsid maturation is highly cooperative and the integrity of the capsid is maintained throughout. The primary mechanism appears to involve the relative rotation of domains in both the outer and inner portions of the major capsid protein. (3) Assembly and Maturation of Bacteriophage Capsids. Our primary interest in bacteriophage capsid assembly lies in the monumental conformational changes that accompany maturation of theirprocapsids. These changes are irreversible, involve partial refolding, and are stringently controlled. As such, they afford unique opportunities for insight into large-scale regulatory conformational changes in protein complexes. Although phages vary widely in size, sequence, and other properties, maturation is a universal feature. We study this event in several systems to exploit expedient aspects of each. We are clarifying the maturation pathway of the HK97 capsid by performing cryo-EM analyses of the earliest precursor, Prohead I, and the penultimate state, Expansion Intermediate III. We are also developing protein display on the dispensable lambda capsid protein, gpD. Results. We visualized Prohead I at 1.1nm resolution and are developing a quasi-atomic model by fitting in the Head II subunit, known from by X-ray crystallography. There are significant movements between the subunit positions occupied in Prohead I and its proteolytically cleaved successor, Prohead II, which we recently characterized by this approach. We have developed a number of fusion proteins in which either gpD or its 15-residue N-terminus are fused to other proteins, and found a way to convert lambda procapsids into mature capsids that lack gpD. Conclusions. These observations imply that processing of Prohead I does not represent a simple excision of density: rather, concomitant adjustments of the inter-subunit interactions take place. Expansion Intermediate III is like Head II in most respects except shape. The last step in maturation amounts to a 4 nm outwards movement of the pentons. We have now assembled the reagents needed to proceed with protein engineering using the lampda capsid as template. (4) Electron Microscopic Studies of HIV- and AIDS-related Proteins. We participate in the NIH Targeted Antiviral Program by pursuing relevant projects in which our expertise in EM-based structural analysis may be applied.(1) Cytopathic Effects of Virus Infection on the Cytoskeleton. HIV infection of human keratinocytes induces ultrastructural changes and massive disruption of microtubules (MT). In FY01, we investigated the interaction of MT in vitro with the HIV-1 transactivator, Rev, and observed rapid depolymerization of MT and Rev filaments, and concomitant appearance of oligomeric rings. Binding of Rev to MT causes pairs of protofilaments to peel off and close into rings. In FY02, we examined the tubulin-Rev rings by cryo-EM and image analysis which revealed Rev-associated density on their inner surface with much greater clarity. We also characterized an alternative form of tubulin ring induced by treatment of MT with the drug, cryptophycin. These rings are smaller and thinner than the Rev-induced ones: they contain 8 dimers circumferentially Cryptophycin binding induces two points of curvature in the protofilaments which are straight or almost so in MT. (2) Structural Characterization of a Ternary Surfactant Phase used to Solubilize Proteins for NMR Spectroscopy. Increasing use is being made of complex media to maintain proteins in a non-aggregated state at suitably high concentrations for NMR. In this context, it is desirable to know the intrinsic structural properties of these media. By direct EM observations, we were able to ascertain that the medium in question is organized as flexible worm-like tubes, ~ 5 nm in diameter. (3) Structural Studies of Hepatitis B Virus and Herpesviruses. HBV shares several key features with HIV. At least two herpesviruses - Kaposi?s Sarcoma-associated Herpesvirus (KSHV) and cytomegalovirus (CMV) - pose significant threats to immunocompromized AIDS patients. Our studies of these viruses (see above) fall partly under the aegis of the IATAP program.